All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Mammalian hematopoietic stem cells develop during embryogenesis and differentiate into the different hematopoietic lineages. After birth, the capacity of myeloid cells to respond to external and/or internal stimuli by the finely tuned production of pro-inflammatory and anti-inflammatory cytokines is gradually acquired, in parallel with the establishment of fully mature lymphocytic immune responses. Interestingly, the responses of both myeloid and lymphoid cell lineages are subject to acetylcholine (ACh) modulation [Kawashima, K., and T. Fujii (2000) Pharmacol. Ther. 86:29-48; Tracey, K. J. (2002) Nature 420:853-9], which involves the α7 nicotinic ACh receptor [Wang, H. et al. (2003) Nature 421:384-8] and are known to be impaired under psychological stress [Miller, G. E. et al. (2002) Health Psychol 21:531-41]. However, the putative protein(s) mediating these developmental and stress-induced processes is yet unknown.
Post-stress leukocytosis, i.e. overproduction of white blood cells (WBC), was first described over 50 years ago. Elevated WBC counts occur after diverse stress insults, e.g. shock, blood loss, in post-partum mothers, following space flight or bacterial infection [Delgado, I. et al. (1994) Gynecol. Obstet. Invest. 38: 227-235; Reizenstein, P. (1979) Br. J. Haematol. 43: 329-334; Stowe, R. P. et al. (1999) J. Leukoc. Biol. 65: 179-186; Toft, P. et al. (1994) Apmis 102: 43-48; Wanahita, A. et al. (2002) Clin. Infect. Dis. 34: 1585-1592]. The initiation of WBC overproduction has been attributed to the elevated serum levels of cortisol, causing both enhanced proliferation and facilitated WBC maturation, predominantly toward the granulocytic lineage [Abramson, N. and Melton, B. (2000) Am. Fam. Physician 62: 2053-2060]. However, the increased levels of cortisol, e.g. following the stressful event of delivery, recede within a few hours [Tuimala, R. et al. (1976) Br. J. Obstet. Gynaecol. 83: 707-710], and cannot account for the prolongation of leukocytosis, especially since the lifespan of granulocytes is extremely short, with 50% of the granulocytes being replaced by the bone marrow daily [Abo, T. and Kawamura, T. (2002) Ther. Apher. 6: 348-357]. The signaling pathways controlling this process therefore remain largely unknown.
Granulocytosis depends upon the production of proinflammatory/hematopoietic cytokines which in peripheral tissues is regulated by acetylcholine (ACh) [Borovikova, L. V. et al. (2000) Nature 405: 458-462; Tracey, K. J. (2002) id ibid.]. Under normal conditions, ACh activates α7 ACh nicotinic receptors on macrophages to attenuate pro-inflammatory cytokine secretion at the post-transcriptional level [Wang, H. (2003) id ibid.]. To determine whether post-stress ACh levels can account for the prolonged granulocytosis effect independently of cortisol, and to delineate the cascade of events that enables this process, the inventors studied circulating acetylcholinesterase (AChE). Agents performing this reaction can further be used to control the production of cytokines in patients with failure of such responses.
Hence, inflammation is an example of inducible hematopoiesis, which occurs whenever there is an increased demand for mature blood cells. Upon activation of the inflammatory response, pro-inflammatory cytokines are secreted by cells of the immune system, and induce accelerated production of hematopoietic cells. Lipopolysaccharide (LPS), the main cell wall component of gram-negative bacteria, is an endotoxin that induces an acute inflammatory response, initiating a signal transduction cascade that leads to the release of inflammatory cytokines, which include tumor necrosis factor (TNF)-α, IL-1β, IL-6 and IL-8. These cytokines activate the mobilization of hematopoietic cells from the bone marrow (BM) and set in motion the migration of leukocytes from blood vessel walls, increasing their numbers in the circulation [Lagasse E, Weissman I L. (1996) J. Immunol. Methods 197:139-150]. The net result of this process is an immediate and dramatic increase in the number of circulating peripheral blood (PB) cells, needed to mount the immune response. This results in a compensatory decrease in cell numbers until more cells are produced in the BM [Nagata Y, et al. (1997) Thromb Haemost. 77:808-814].
Many factors are involved in abating the inflammatory response allowing hemostasis to return. Acetylcholine (ACh), is one of the recently discovered factors that attenuates the pro-inflammatory cytokine secretion by activating nicotinic receptors on macrophages at the post-transcriptional level [Wang H. et al. (2003) Nature 421:384-388]. Circulating acetylcholinesterase (AChE) controls the levels of ACh, suggesting promotion of the inflammatory process under AChE excess [Pick M. et al. (2004) Ann. NY Acad. Sci. 1018:85-95]. AChE has three variant forms,—Synaptic (S), Erythrocytic (E) and Readthrough (R), is ubiquitously expressed in hematopoietic cell lineages especially in megakaryocytes (Mks) and erythrocytes [Kawashima K, and Fujii T. (2000) Pharmacol. Ther. 86:29-48; Lev-Lehman E. et al. (1997) Blood 89:3644-3653; Grisaru D. et al. (2001) Molecular Medicine 7:93-105] and is thought to be a potential growth factor for hematopoiesis [Grisaru (2001) id ibid.; Deutsch V. et al. (2002) Exp. Hematol. 30:1153-1161].
AChE-R is expressed in multiple embryonic and tumor cells, where it displays morphogenic functions, but it is rarely found in healthy and unstressed adult tissues [Grisaru (1999a) id ibid.; Karpel (1994) id ibid.; Soreq, H. and S. Seidman (2001) Nat. Rev. Neurosci. 2:294-302; Grisaru, D. et al. (1999b) Mol. Cell. Biol. 19:788-795] or human sera [Brenner et al. (2003) FASEB J. 17(2):214-22]. Cortisol induces AChE-R production in cultured CD34+ blood cell progenitors [Grisaru (2001) id ibid.], while ARP26, a synthetic peptide designed to mimic the cleavable C-terminal sequence of AChE-R, promotes hematopoietic proliferation in vitro [Grisaru (2001) id ibid.].
The up regulation of AChE expression during megakaryopoiesis was initially reported in rats, where the fraction of AChE-positive BM cells increased following induction of thrombocytopenia [Jackson C W. (1973) Blood 42:413-421]. Functional involvement of this enzyme was indicated by suppression of AChE synthesis, which induced transient decreases in murine megakaryocyte progenitors [Lev-Lehman (1997) id ibid.].
Platelet production is a self-regulated process primarily induced by thrombocytopenia where a drastic reduction in platelets stimulates the production of thrombopoietin (TPO). Subsequently, as platelet counts return to normal, TPO is effectively cleared from the circulation, by means of binding to its receptor, c-mpl, and uptake into platelets and megakaryocytes. TPO is the main physiological growth factor for megakaryocyte proliferation, differentiation and platelet production. Nevertheless, c-mpl−/− and TPO−/− knockout mice have a residual 10% of normally functioning megakaryocytes and platelets, which cannot be attributed to IL-6, IL-11 or leukemia inhibitory factor (LIF), which are also known to induce megakaryocyte differentiation [Ishibashi T. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5953-5957; Teramura M., et al. (1996) Cancer Chemother. Pharmacol. 38:Suppl:S99-102; Nakashima K. et al. (1998) Semin. Hematol. 35: 210-221; Gainsford T. et al. (2000) Blood 95: 528-534] suggesting the involvement of other factor(s) in this process.
Pancytopenia and prolonged thrombocytopenia are significant clinical problems for patients undergoing BM transplantation. Engraftment of transplanted BM is usually accomplished within 2 to 3 weeks, during which period the patient is susceptible to life-threatening infections and bleeding. Platelet recovery after autologous stem cells or cord blood (CB) transplantation is significantly delayed (up to 6 weeks post transplant) due to lack of sufficient megakaryocyte precursors in the grafts. The paucity of megakaryocyte progenitor cells in grafts, and not inferior levels of TPO, is the cause for delayed platelet recovery observed post cord blood and autologous transplantation [Kuter D. J. (2002) Transfusion 42:279-283; Kanamaru S. et al. (2000) Stem Cells 18:190-195].
Thus, within their individual microenvironment, blood cells receive a plethora of external stimuli which influence transcription and processing of many reactive molecules. Particular alternatively spliced AChE variants may be candidates to exert both enzymatic and non-catalytic functions on these cells. The expression of AChE-S in blood cells has been associated with terminal differentiation [Chan, R. Y. Y. et al. (1998) J. Biol. Chem. 273:9727-9733] and apoptosis [Zhang, X. J. et al. (2002) Cell Death Differ. 9:790-800]. In contrast, AChE-R and the synthetic peptide ARP were associated with stem myeloid cell proliferation [Grisaru (2001) id ibid.; Deutsch et al. (2002) id ibid.].
The present inventors performed a comprehensive study to correctly evaluate the potential contribution of AChE towards differentiation, proliferative or apoptotic events in hematopoiesis, and in inflammatory responses under stress stimuli, specific variants were identified, their levels quantified and their subcellular localization (i.e. on the cell surface and/or intracellular) determined in specific blood cell lineages.
The inventors considered, as a working hypothesis, circulating AChE-R to be a modulator of sustained granulocytosis effects in hematopoietic progenitors. To find out whether AChE-R and/or ARP are associated with post-stress granulocytosis and cytokine production, the inventors initiated a study aimed at delineating the in vivo and ex vivo regulation of AChE-R production in stress-induced myelopoietic processes.
Thus, an aim of the present invention is to provide novel uses for an AChE-derived peptide, as an agent capable of inducing granulopoiesis, as demonstrated in the following Examples.
It is another aim of the present invention to provide a method for the treatment of conditions that induce a low granulocytic cell count, administering said AChE-derived peptide, and compositions thereof, to a subject in need.
Further, the present invention provides methods of evaluating lymphocytic activity, based on the expression of the different AChE forms on lymphocytes.
Other purposes and advantages of the invention will appear as the description proceeds.